Advancement of capture immunoassay for real-time monitoring of hepatitis E virus-infected monkey

Highlights

• In situ silver deposition on surface of gold nanoparticles was prepared.

• An advanced platform for immunoassay has amplified signal significantly.

• HEV was successfully detected from fecal sample from HEV-infected monkey.

• Sensor performance was correlated with their standard RT-qPCR data.

Abstract

Rapid increasing outbreak of Hepatitis E virus (HEV) shows an urgent need of HEV detection. Instead of time consuming and expensive RT-qPCR, an efficient and quick monitoring system is in utmost demand which can be comparable with the RT-qPCR in term of reliability and detection limit. An advanced platform for immunoassay has been constructed in this study by a nanozyme that constitutes anti-HEV IgG antibody-conjugated gold nanoparticles (Ab-AuNPs) as core and in situ silver deposition on the surface of Ab-AuNPs as outer shell. The virus has been entrapped on the nanocomposites while the silver-shell has decomposed back to the silver ions (Ag⁺) by adding a tetramethylbenzidine (TMBZ) and hydrogen peroxide (H₂O₂) which indirectly quantifies the target virus concentration. Counterpart to only applying nanozyme, by incorporation of the enhanced effect of Ag shell on the AuNP-based nanozyme, the advance deposition has been confirmed to prove the signal amplification mechanism in the proposed immunoassay. Most importantly, the sensor performances have examined on the HEV, collected from the HEV-infected monkey over a period of 45 days. It was successfully correlated with the standard RT-qPCR data, showing the applicability of this immunoassay as a real-time monitoring on the HEV infection. The in situ formation of AuNPs@Ag as nanozyme in this capture immunoassay leads to a promising advancement over the conventional methods and nanozyme-based immunoassay in real application which can be a good substitute of RT-qPCR in near future.

Introduction

HEV infection has become a serious health concern due to the increasing outbreak of Hepatitis E in developing as well as developed countries, which are mostly contributed via waterborne and zootomic-transmitted pathway [1]. Hepatitis E is associated to liver failure and acute liver disease, with higher fatality during pregnancy [2,3]. Recently, major development in HEV detection platform is targeting on the RNA-targeting RT-PCR and antibodies (IgG, IgM and IgA) that were produced after the infection of HEV [4,5]. Due to the limited available diagnostic assay on HEV, a reliable antigen-targeting immunoassay is required to outstand the emerging outbreak by early detection of the disease agent. Instead of RT-qPCR, enzyme-linked immunosorbent assay (ELISA) has been well-known for its simplicity with step-by-step procedure to determine the presence of the target analyte [6,7]. Commonly, a colorimetric-based ELISA utilized peroxidase as the enzyme which catalyzes the oxidation of chromogen substrate by hydrogen peroxide. However, its low sensitivity has been addressed as a strong limitation in detecting HEV type of viral agents, where very low concentration of virus can make the fatality [6]. This issue can be addressed by the signal amplification used in the platform [8,9]. An upgraded ELISA-based biosensor with comparable reliability of RT-qPCR has been in necessity to restrict the outbreak of HEV.

Since the first discovery of Fe₃O₄ nanoparticles (NPs) exhibiting peroxidase-like activity, known as nanozyme, more application with various kind of nanozymes has been discovered, especially in biosensing technology [[9], [10], [11]]. Various applications such as enzymatic activity [12], nanozymes has been shown to either enhance the activity of the coupled enzymes or exhibiting robust enzymatic-like activity [13,14]. Nanomaterials, especially noble metals, such as gold (Au) [15], silver (Ag) [16] and platinum (Pt) [17], have been employed to substitute the current enzyme used in the immunoassay due to their intrinsic peroxidase-like activity, good stability and biocompatibility [[18], [19], [20], [21]]. Interestingly, hybrid nanomaterial, such as graphene/gold nanohybrid [22], gold-decorated organic framework [23], and bimetallic [[24], [25], [26], [27]] to trimetallic [28,29] nanostructure, demonstrated unique morphology and increasing catalytic activity significantly [30]. To achieve the desired specificity, proper bioconjugation has to be applied successfully. However, in contrast, to maintain the specificity of the nanozyme, bioconjugation can reduce its catalytical activity to some extent [20,31]. To make an optimized condition for modified immunoassays, noble metals have been emerging as a potential platform for easy bioconjugation step with compromising their activity. In this case, Au can be an automatic choice for its less toxicity and cost effectiveness than Pt, and higher plasmonic characteristic compared to Ag in addition with its easy functionalized and control-synthesized properties [32].

In order to develop a highly sensitive nanozyme-based immunoassay for HEV detection, in this study, a signal amplification strategy has been introduced by utilizing silver deposition on Au nanozyme. In our previous study, a proposed immunoassay was demonstrated for norovirus detection and enhanced colorimetric signal in low concentration [20]. Therefore, in this study, more reliable approach was applied to on-site detection and the results were compared to conventional RT-qPCR. However, similar with most of the colorimetric assays, the bare gold nanoparticles (AuNPs) could not be able to perform well in the desired concentration range for real sample analysis. To enhance the performance and stability in real sample medium, in this work, we have proposed a core shell nanocomposite with silver. Silver deposition on gold nanoparticles, was already previously reported for its application in surface-enhanced Raman scattering detection [33], mass spectrometry [34], and opacity densitometry [35], however its applicability is not well explored in the field of catalytic enhancement, especially for virus detection. According to our hypothesis, the AuNPs core with Ag shell structure (AuNPs@Ag) has been introduced for the enhanced catalytic activity of the Ag towards TMB-based color development. The homogeneous distributed AuNPs@Ag with higher stability have been successfully combined to make an upgraded platform for virus detection, especially for real sample analysis. To establish the mechanism, the AuNPs@Ag nanocomposite was synthesized separately and optimized to get the best suited condition for the detection. Then the system was applied in situ deposition during virus-sensing process in the antigen-antibody interaction. A capture immunoassay was demonstrated for HEV detection by using HEV-like particles (HEV-LPs) as the model. By conjugating the Au nanozyme with anti-HEV IgG antibody, the proposed immunoassay demonstrated an amplified signal for low concentration of HEV-LPs. In the presence of silver deposition solution containing silver ions (Ag⁺) and hydroquinone (HQ) and chromogen solution containing hydrogen peroxide (H₂O₂) and 3,3′,5,5′-tetramethylbenzidine (TMBZ), the color as a detection signal was significantly amplified compared to the bare AuNP system. In the final step, the nanozyme-based capture immunoassay was challenged to detect the viral HEV containing in fecal samples collected from HEV-infected monkey and showed a comparable trend with RT-qPCR, representing its promising application towards the detection of HEV.